5.1 Soil toxicity tests provide information concerning the toxicity and bioavailability of chemicals associated with soils to terrestrial organisms. As important members of the soil fauna, nematodes have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Bacterial-feeding nematodes such as C. elegans feed on soil microbes and contribute to the breakdown of organic matter. They are also of extreme importance in the cycling and degradation of key nutrients in soil ecosystems (9). Soil nematodes also serve as a source of prey and nutrients for fauna and microflora such as soil nematophagous fungi (10). A major change in the abundance of soil invertebrates such as nematodes, either as a food source or as organisms functioning properly in trophic energy transfer and nutrient cycling, could have serious adverse ecological effects on the entire terrestrial system.5.2 Results from soil tests might be an important consideration when assessing the hazards of materials to terrestrial organisms.5.3 The soil test might be used to determine the temporal or spatial distribution of soil toxicity. Test methods can be used to detect horizontal and vertical gradients in toxicity.5.4 Results of soil tests could be used to compare the sensitivities of different species.5.5 An understanding of the effect of these parameters on toxicity may be gained by varying soil characteristics such as pH, clay content, and organic material.5.6 Results of soil tests may be useful in helping to predict the effects likely to occur with terrestrial organisms in field situations.5.6.1 Field surveys can be designed to provide either a qualitative or quantitative evaluation of biological effects within a site or among sites.5.6.2 Soil surveys evaluating biological effects are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic conditions. Statistical correlation can be improved and costs reduced if subsamples of soil for laboratory tests, geochemical analyses, and community structure are taken simultaneously from the same grab of the same site.5.7 Soil toxicity tests can be an important tool for making decisions regarding the extent of remedial action necessary for contaminated terrestrial sites.1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of chemicals associated with soil to nematodes from soil toxicity tests. This standard is based on a modification to Guide E1676. The methods are designed to assess lethal or sublethal toxic effects on nematodes in short-term tests in terrestrial systems. Soils to be tested may be (1) references soils or potentially toxic soil sites; (2) artificial, reference, or site soils spiked with compounds; (3) site soils diluted with reference soils; or (4) site or reference soils diluted with artificial soil. Test procedures are described for the species Caenorhabditis elegans (see Annex A1). Methods described in this guide may also be useful for conducting soil toxicity tests with other terrestrial species, although modifications may be necessary.1.2 Summary of Previous Studies—Initial soil toxicity testing using the free-living, bacterivorous soil nematode Caenorhabditis elegans was developed by Donkin and Dusenbery (1).2 Following the development of an effective method of recovery of C. elegans from test soils, the organism was used to identify factors that affect the toxicity of zinc, cadmium, copper, and lead (2) . Freeman et al. further refined the nematode bioassay by decreasing the quantity of soil and spiking solution volumes, determining test acceptability criteria, and developing control charts to assess worm health using copper as a reference toxicant (3). More recently, the toxicological effects of nitrate and chloride metallic salts in two natural soils were compared (4) . LC50 values for C. elegans exposed for 24-h to nitrate salts of cadmium, copper, zinc, lead and nickel in an artificial soil (see Annex A2) were found to be similar to LC50 values for the earthworm, Eisenia fetida (5). Increasing the exposure time to 48-h resulted in much lower LC50 values (6). However, longer exposure times necessitate the addition of food and lead to lower recovery percentages in soils high in organic matter. A modification of the recovery method has also been used with a transgenic strain of C. elegans used as a soil biomonitoring tool to assess sub-lethal effects of metal exposures in soil (7) . A variety of sub-lethal endpoints have been developed using C. elegans in aquatic media and may prove useful for assessing soil exposures (8).1.3 Modification of these procedures might be justified by special needs. The results of tests conducted using typical procedures may not be comparable to results using this guide. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting soil toxicity tests with terrestrial worms.1.4 The results from field-collected soils used in toxicity tests to determine a spatial or temporal distribution of soil toxicity may be reported in terms of the biological effects on survival or sublethal endpoints. These procedures can be used with appropriate modifications to conduct soil toxicity tests when factors such as temperature, pH, and soil characteristics (for example, particle size, organic matter content, and clay content) are of interest or when there is a need to test such materials as sewage sludge. These methods might also be useful for conducting bioaccumulation tests.1.5 The results of toxicity tests with (1) materials (for example, chemicals or waste mixtures) added experimentally to artificial soil, reference soils, or site soils, (2) site soils diluted with reference soils, and (3) site or reference soils diluted with artificial soil, so as to create a series of concentrations, may be reported in terms of an LC50 (median lethal concentration) and sometimes an EC50 (median effect concentration).1.6 This guide is arranged as follows: 1Referenced Documents 2Terminology 3Summary of Guide 4 5Interferences 6Apparatus 7Safety Precautions 8Soil 9Test Organism 10Procedure 11Analytical Methodology 12Acceptability of Test 13Calculation of Results 14Report 15Annexes A1. Caenorhabditis elegans  A2. Artificial Soil CompositionReferences  1.7 The values stated in SI units are to be regarded as the standard.1.8 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. While some safety considerations are included in this guide, it is beyond the scope of this standard to encompass all safety requirements necessary to conduct soil toxicity tests. Specific precautionary statements are given in Section 8.1.9 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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This test method provides a rapid means of determining the acute toxicity of an aqueous waste, or waste extract, prior to and following biological treatment, and contributes to assessing the potential biodegradability of the waste (see 1.1, 1.2, and Note 1). The change in toxicity to the marine bacterium P. phosphoreum with respect to time may serve as an indication of the biodegradation potential. Sample analyses are usually obtained in 45 to 60 min, with as little as 5 mL of sample required (5). Samples with high suspended solids concentrations may test nontoxic to the bacteria, while still exhibiting significant toxicity to freshwater organisms, due to those suspended solids. The absorbance correction procedure included in this test method allows for the analysis of highly colored lightabsorbing samples, by providing a means for mathematically adjusting the light output readings to account for light lost due to absorption. 1.1 This test method (1) covers a procedure for the rapid evaluation of the toxicity of wastewaters and aqueous extracts from contaminated soils and sediments, to the luminescent marine bacterium Photobacterium phosphoreum, prior to and following biological treatment. This test method is meant for use as a means to assess samples resulting from biotreatability studies. Sensitivity data for P. phosphoreum to over 1300 chemicals have been reported in the literature (2). Some of the publications are very relevant to this test method (3). The data obtained from this test method, when combined with respirometry, total organic carbon (TOC), biochemical oxygen demand (BOD), chemical oxygen demand (COD), or spectrophotometric data, can assist in the determination of the degree of biodegradability of a contaminant in water, soil, or sediment (3). The percentage difference between the IC20 of treated and untreated sample is used to assess the progress of detoxification. 1.2 This test method is applicable to the evaluation of the toxicity (to a specific microbe) and its implication on the biodegradation of aqueous samples from laboratory research bio-reactors (liquid or soil), pilot-plant biological treatment systems, full-scale biological treatment systems, and land application processes (see Notes 1 and 2). Note 1—If the biologically treated material is to be discharged in such a manner as to potentially impact surface waters and ground water, or both, then the user must consult appropriate regulatory guidance documents to determine the proper test species for evaluating potential environmental impact (4). Correlations between data concerning reduction in toxicity produced by this test method and by procedures for acute or short-term chronic toxicity tests, or both, utilizing invertebrates and fish (see Guides E729 and E1192E729E1192), should be established, wherever possible. Note 2—Color (especially red and brown), turbidity, and suspended solids interfere with this test method by absorbing or reflecting light. In these situations data are corrected for these effects by use of an absorbance correction procedure included in this test method (see 5.3, 6.1, and 6.2). 1.3 The results of this test method are reported in terms of an inhibitory concentration (IC), which is the calculated concentration of sample required to produce a specific quantitative and qualitative inhibition. The inhibition measured is the quantitative reduction in light output of luminescent marine bacteria (that is, IC20 represents the calculated concentration of sample that would produce a 20 % reduction in the light output of exposed bacteria over a specified time). 1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only. 1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use. Specific hazard statements are given in Section 9.

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1.1 This practice covers a standard procedure for preparing the residual solids obtained at the end of standard methods for biodegradability testing of plastics in solid waste, for subsequent toxicity and compost quality testing. The practice yields mixtures that can be used as such for terrestrial toxicity testing or that can be submitted to water extraction for further aquatic toxicity tests, in accordance with Practice D 5152, and in conjunction with Method D 4229, Guides E 729, E 1192, E 1295, and E 1440, or other currently accepted toxicity test methods (see OECD Guidelines 201, 202, 203, 207, and 208 or U.S. EPA 40FR797A, as well as other documents such as A New Manual for Conducting Microtox Test with the Model 500 Analyzer the work on cyst-based toxicity tests by Centeno, et al). The mixtures can also be used for further soil contact biodegradation testing.1.2 This practice provides for storage and drying of the mixtures obtained at the end of the test methods for determination of the biodegradability of plastics under controlled composting conditions (Test Method D 5338), and under high-solids anaerobic digestion (Test Method D 5511). The mixtures contain the biologically decomposed residuals from solid waste and from the plastic materials. For the blanks, the residuals will be derived only from the biologically decomposed solid waste. In the event that a particular sample does not pass the toxicity test, chemical characterization of the degradation products can be performed on the sample to determine the source of the toxicity. Description of the performance of these analyses is beyond the scope of this practice.1.3 There is no ISO standard that is equivalent to this practice.1.4 The values stated in SI units are to be regarded as the standard. The values given in parentheses are for information only.1.5 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety and health practices and determine the applicability of regulatory limitations prior to use.

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5.1 General: 5.1.1 Sediment provides habitat for many aquatic organisms and is a major repository for many of the more persistent chemicals that are introduced into surface waters. In the aquatic environment, most anthropogenic chemicals and waste materials including toxic organic and inorganic chemicals eventually accumulate in sediment. Mounting evidences exists of environmental degradation in areas where USEPA Water Quality Criteria (WQC; Stephan et al.(66)) are not exceeded, yet organisms in or near sediments are adversely affected Chapman, 1989 (67). The WQC were developed to protect organisms in the water column and were not directed toward protecting organisms in sediment. Concentrations of contaminants in sediment may be several orders of magnitude higher than in the overlying water; however, whole sediment concentrations have not been strongly correlated to bioavailability Burton, 1991 (68). Partitioning or sorption of a compound between water and sediment may depend on many factors including: aqueous solubility, pH, redox, affinity for sediment organic carbon and dissolved organic carbon, grain size of the sediment, sediment mineral constituents (oxides of iron, manganese, and aluminum), and the quantity of acid volatile sulfides in sediment Di Toro et al. 1991(69) Giesy et al. 1988 (70). Although certain chemicals are highly sorbed to sediment, these compounds may still be available to the biota. Chemicals in sediments may be directly toxic to aquatic life or can be a source of chemicals for bioaccumulation in the food chain.5.1.2 The objective of a sediment test is to determine whether chemicals in sediment are harmful to or are bioaccumulated by benthic organisms. The tests can be used to measure interactive toxic effects of complex chemical mixtures in sediment. Furthermore, knowledge of specific pathways of interactions among sediments and test organisms is not necessary to conduct the tests Kemp et al. 1988, (71). Sediment tests can be used to: (1) determine the relationship between toxic effects and bioavailability, (2) investigate interactions among chemicals, (3) compare the sensitivities of different organisms, (4) determine spatial and temporal distribution of contamination, (5) evaluate hazards of dredged material, (6) measure toxicity as part of product licensing or safety testing, (7) rank areas for clean up, and (8) estimate the effectiveness of remediation or management practices.5.1.3 A variety of methods have been developed for assessing the toxicity of chemicals in sediments using amphipods, midges, polychaetes, oligochaetes, mayflies, or cladocerans (Test Method E1706, Guide E1525, Guide E1850; Annex A1, Annex A2; USEPA, 2000 (72), EPA 1994b, (73), Environment Canada 1997a, (74), Enviroment Canada 1997b,(75)). Several endpoints are suggested in these methods to measure potential effects of contaminants in sediment including survival, growth, behavior, or reproduction; however, survival of test organisms in 10-day exposures is the endpoint most commonly reported. These short-term exposures that only measure effects on survival can be used to identify high levels of contamination in sediments, but may not be able to identify moderate levels of contamination in sediments (USEPA USEPA, 2000 (72); Sibley et al.1996, (76); Sibley et al.1997a, (77); Sibley et al.1997b, (78); Benoit et al.1997, (79); Ingersoll et al.1998, (80)). Sublethal endpoints in sediment tests might also prove to be better estimates of responses of benthic communities to contaminants in the field, Kembel et al. 1994 (81). Insufficient information is available to determine if the long-term test conducted with Leptocheirus plumulosus (Annex A2) is more sensitive than 10-d toxicity tests conducted with this or other species.5.1.3.1 The decision to conduct short-term or long-term toxicity tests depends on the goal of the assessment. In some instances, sufficient information may be gained by measuring sublethal endpoints in 10-day tests. In other instances, the 10-day tests could be used to screen samples for toxicity before long-term tests are conducted. While the long-term tests are needed to determine direct effects on reproduction, measurement of growth in these toxicity tests may serve as an indirect estimate of reproductive effects of contaminants associated with sediments (Annex A1).5.1.3.2 Use of sublethal endpoints for assessment of contaminant risk is not unique to toxicity testing with sediments. Numerous regulatory programs require the use of sublethal endpoints in the decision-making process (Pittinger and Adams, 1997, (82)) including: (1) Water Quality Criteria (and State Standards); (2) National Pollution Discharge Elimination System (NPDES) effluent monitoring (including chemical-specific limits and sublethal endpoints in toxicity tests); (3) Federal Insecticide, Rodenticide and Fungicide Act (FIFRA) and the Toxic Substances Control Act (TSCA, tiered assessment includes several sublethal endpoints with fish and aquatic invertebrates); (4) Superfund (Comprehensive Environmental Responses, Compensation and Liability Act; CERCLA); (5) Organization of Economic Cooperation and Development (OECD, sublethal toxicity testing with fish and invertebrates); (6) European Economic Community (EC, sublethal toxicity testing with fish and invertebrates); and (7) the Paris Commission (behavioral endpoints).5.1.4 Results of toxicity tests on sediments spiked at different concentrations of chemicals can be used to establish cause and effect relationships between chemicals and biological responses. Results of toxicity tests with test materials spiked into sediments at different concentrations may be reported in terms of an LC50 (median lethal concentration), an EC50 (median effect concentration), an IC50 (inhibition concentration), or as a NOEC (no observed effect concentration) or LOEC (lowest observed effect concentration). However, spiked sediment may not be representative of chemicals associated with sediment in the field. Mixing time Stemmer et al. 1990b, (83), aging ( Landrum et al. 1989, (84), Word et al. 1987, (85), Landrum et al., 1992,(86)), and the chemical form of the material can affect responses of test organisms in spiked sediment tests.5.1.5 Evaluating effect concentrations for chemicals in sediment requires knowledge of factors controlling their bioavailability. Similar concentrations of a chemical in units of mass of chemical per mass of sediment dry weight often exhibit a range in toxicity in different sediments Di Toro et al. 1990, (87) Di Toro et al. 1991,(69). Effect concentrations of chemicals in sediment have been correlated to interstitial water concentrations, and effect concentrations in interstitial water are often similar to effect concentrations in water-only exposures. The bioavailability of nonionic organic compounds in sediment is often inversely correlated with the organic carbon concentration. Whatever the route of exposure, these correlations of effect concentrations to interstitial water concentrations indicate that predicted or measured concentrations in interstitial water can be used to quantify the exposure concentration to an organism. Therefore, information on partitioning of chemicals between solid and liquid phases of sediment is useful for establishing effect concentrations Di Toro et al. 1991, (69).5.1.6 Field surveys can be designed to provide either a qualitative reconnaissance of the distribution of sediment contamination or a quantitative statistical comparison of contamination among sites.5.1.7 Surveys of sediment toxicity are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic data. Statistical correlations may be improved and sampling costs may be reduced if subsamples are taken simultaneously for sediment tests, chemical analyses, and benthic community structure.5.1.8 Table 2 lists several approaches the USEPA has considered for the assessment of sediment quality USEPA, 1992, (88). These approaches include: (1) equilibrium partitioning, (2) tissue residues, (3) interstitial water toxicity, (4) whole-sediment toxicity and sediment-spiking tests, (5) benthic community structure, (6) effect ranges (for example, effect range median, ERM), and (7) sediment quality triad (see USEPA, 1989a, 1990a, 1990b and 1992b, (89, 90, 91, 92 and Wenning and Ingersoll (2002 (93)) for a critique of these methods). The sediment assessment approaches listed in Table 2 can be classified as numeric (for example, equilibrium partitioning), descriptive (for example, whole-sediment toxicity tests), or a combination of numeric and descriptive approaches (for example, ERM, USEPA, 1992c, (94). Numeric methods can be used to derive chemical-specific sediment quality guidelines (SQGs). Descriptive methods such as toxicity tests with field-collected sediment cannot be used alone to develop numerical SQGs for individual chemicals. Although each approach can be used to make site-specific decisions, no one single approach can adequately address sediment quality. Overall, an integration of several methods using the weight of evidence is the most desirable approach for assessing the effects of contaminants associated with sediment, (Long et al. 1991(95) MacDonald et al. 1996 (96) Ingersoll et al. 1996 (97) Ingersoll et al. 1997 (98), Wenning and Ingersoll 2002 (93)). Hazard evaluations integrating data from laboratory exposures, chemical analyses, and benthic community assessments (the sediment quality triad) provide strong complementary evidence of the degree of pollution-induced degradation in aquatic communities (Burton, 1991 (68), Chapman 1992, 1997 (99, 100).)5.2 Regulatory Applications—Test Method E1706 provides information on the regulatory applications of sediment toxicity tests.5.3 Performance-based Criteria: 5.3.1 The USEPA Environmental Monitoring Management Council (EMMC) recommended the use of performance-based methods in developing standards, (Williams, 1993 (101). Performance-based methods were defined by EMMC as a monitoring approach which permits the use of appropriate methods that meet preestablished demonstrated performance standards (11.2).5.3.2 The USEPA Office of Water, Office of Science and Technology, and Office of Research and Development held a workshop to provide an opportunity for experts in the field of sediment toxicology and staff from the USEPA Regional and Headquarters Program offices to discuss the development of standard freshwater, estuarine, and marine sediment testing procedures (USEPA, 1992a, 1994a (88, 102)). Workgroup participants arrived at a consensus on several culturing and testing methods. In developing guidance for culturing test organisms to be included in the USEPA methods manual for sediment tests, it was agreed that no one method should be required to culture organisms. However, the consensus at the workshop was that success of a test depends on the health of the cultures. Therefore, having healthy test organisms of known quality and age for testing was determined to be the key consideration relative to culturing methods. A performance-based criteria approach was selected in USEPA, 2000 (72) as the preferred method through which individual laboratories could use unique culturing methods rather than requiring use of one culturing method.5.3.3 This standard recommends the use of performance-based criteria to allow each laboratory to optimize culture methods and minimize effects of test organism health on the reliability and comparability of test results. See Annex A1 and Annex A2 for a listing of performance criteria for culturing or testing.1.1 This test method covers procedures for testing estuarine or marine organisms in the laboratory to evaluate the toxicity of contaminants associated with whole sediments. Sediments may be collected from the field or spiked with compounds in the laboratory. General guidance is presented in Sections 1 – 15 for conducting sediment toxicity tests with estuarine or marine amphipods. Specific guidance for conducting 10-d sediment toxicity tests with estuarine or marine amphipods is outlined in Annex A1 and specific guidance for conducting 28-d sediment toxicity tests with Leptocheirus plumulosus is outlined in Annex A2.1.2 Procedures are described for testing estuarine or marine amphipod crustaceans in 10-d laboratory exposures to evaluate the toxicity of contaminants associated with whole sediments (Annex A1; USEPA 1994a (1)). Sediments may be collected from the field or spiked with compounds in the laboratory. A toxicity method is outlined for four species of estuarine or marine sediment-burrowing amphipods found within United States coastal waters. The species are Ampelisca abdita, a marine species that inhabits marine and mesohaline portions of the Atlantic coast, the Gulf of Mexico, and San Francisco Bay; Eohaustorius estuarius, a Pacific coast estuarine species; Leptocheirus plumulosus, an Atlantic coast estuarine species; and Rhepoxynius abronius , a Pacific coast marine species. Generally, the method described may be applied to all four species, although acclimation procedures and some test conditions (that is, temperature and salinity) will be species-specific (Sections 12 and Annex A1). The toxicity test is conducted in 1-L glass chambers containing 175 mL of sediment and 775 mL of overlying seawater. Exposure is static (that is, water is not renewed), and the animals are not fed over the 10-d exposure period. The endpoint in the toxicity test is survival with reburial of surviving amphipods as an additional measurement that can be used as an endpoint for some of the test species (for R. abronius and E. estuarius). Performance criteria established for this test include the average survival of amphipods in negative control treatment must be greater than or equal to 90 %. Procedures are described for use with sediments with pore-water salinity ranging from >0 o/oo to fully marine.1.3 A procedure is also described for determining the chronic toxicity of contaminants associated with whole sediments with the amphipod Leptocheirus plumulosus in laboratory exposures (Annex A2; USEPA-USACE 2001(2)). The toxicity test is conducted for 28 d in 1-L glass chambers containing 175 mL of sediment and about 775 mL of overlying water. Test temperature is 25° ± 2 °C, and the recommended overlying water salinity is 5 o/oo ± 2 o/oo (for test sediment with pore water at 1 o/oo to 10 o/oo ) or 20 o/oo ± 2 o/oo (for test sediment with pore water >10 o/oo ). Four hundred millilitres of overlying water is renewed three times per week, at which times test organisms are fed. The endpoints in the toxicity test are survival, growth, and reproduction of amphipods. Performance criteria established for this test include the average survival of amphipods in negative control treatment must be greater than or equal to 80 % and there must be measurable growth and reproduction in all replicates of the negative control treatment. This test is applicable for use with sediments from oligohaline to fully marine environments, with a silt content greater than 5 % and a clay content less than 85 %.1.4 A salinity of 5 or 20 o/oo is recommended for routine application of 28-d test with L. plumulosus (Annex A2; USEPA-USACE 2001 (2)) and a salinity of 20 o/oo is recommended for routine application of the 10-d test with E. estuarius or L. plumulosus (Annex A1). However, the salinity of the overlying water for tests with these two species can be adjusted to a specific salinity of interest (for example, salinity representative of site of interest or the objective of the study may be to evaluate the influence of salinity on the bioavailability of chemicals in sediment). More importantly, the salinity tested must be within the tolerance range of the test organisms (as outlined in Annex A1 and Annex A2). If tests are conducted with procedures different from those described in 1.3 or in Table A1.1 (for example, different salinity, lighting, temperature, feeding conditions), additional tests are required to determine comparability of results (1.10). If there is not a need to make comparisons among studies, then the test could be conducted just at a selected salinity for the sediment of interest.1.5 Future revisions of this standard may include additional annexes describing whole-sediment toxicity tests with other groups of estuarine or marine invertebrates (for example, information presented in Guide E1611 on sediment testing with polychaetes could be added as an annex to future revisions to this standard). Future editions to this standard may also include methods for conducting the toxicity tests in smaller chambers with less sediment (Ho et al. 2000 (3), Ferretti et al. 2002 (4)).1.6 Procedures outlined in this standard are based primarily on procedures described in the USEPA (1994a (1)), USEPA-USACE (2001(2)), Test Method E1706, and Guides E1391, E1525, E1688, Environment Canada (1992 (5)), DeWitt et al. (1992a (6); 1997a (7)), Emery et al. (1997 (8)), and Emery and Moore (1996 (9)), Swartz et al. (1985 (10)), DeWitt et al. (1989 (11)), Scott and Redmond (1989 (12)), and Schlekat et al. (1992 (13)).1.7 Additional sediment toxicity research and methods development are now in progress to (1) refine sediment spiking procedures, (2) refine sediment dilution procedures, (3) refine sediment Toxicity Identification Evaluation (TIE) procedures, (4) produce additional data on confirmation of responses in laboratory tests with natural populations of benthic organisms (that is, field validation studies), and (5) evaluate relative sensitivity of endpoints measured in 10- and 28-d toxicity tests using estuarine or marine amphipods. This information will be described in future editions of this standard.1.8 Although standard procedures are described in Annex A2 of this standard for conducting chronic sediment tests with L. plumulosus, further investigation of certain issues could aid in the interpretation of test results. Some of these issues include further investigation to evaluate the relative toxicological sensitivity of the lethal and sublethal endpoints to a wide variety of chemicals spiked in sediment and to mixtures of chemicals in sediments from contamination gradients in the field (USEPA-USACE 2001 (2)). Additional research is needed to evaluate the ability of the lethal and sublethal endpoints to estimate the responses of populations and communities of benthic invertebrates to contaminated sediments. Research is also needed to link the toxicity test endpoints to a field-validated population model of L. plumulosus that would then generate estimates of population-level responses of the amphipod to test sediments and thereby provide additional ecologically relevant interpretive guidance for the laboratory toxicity test.1.9 This standard outlines specific test methods for evaluating the toxicity of sediments with A. abdita, E. estuarius, L. plumulosus, and R. abronius. While standard procedures are described in this standard, further investigation of certain issues could aid in the interpretation of test results. Some of these issues include the effect of shipping on organism sensitivity, additional performance criteria for organism health, sensitivity of various populations of the same test species, and confirmation of responses in laboratory tests with natural benthos populations.1.10 General procedures described in this standard might be useful for conducting tests with other estuarine or marine organisms (for example, Corophium spp., Grandidierella japonica, Lepidactylus dytiscus, Streblospio benedicti), although modifications may be necessary. Results of tests, even those with the same species, using procedures different from those described in the test method may not be comparable and using these different procedures may alter bioavailability. Comparison of results obtained using modified versions of these procedures might provide useful information concerning new concepts and procedures for conducting sediment tests with aquatic organisms. If tests are conducted with procedures different from those described in this test method, additional tests are required to determine comparability of results. General procedures described in this test method might be useful for conducting tests with other aquatic organisms; however, modifications may be necessary.1.11 Selection of Toxicity Testing Organisms: 1.11.1 The choice of a test organism has a major influence on the relevance, success, and interpretation of a test. Furthermore, no one organism is best suited for all sediments. The following criteria were considered when selecting test organisms to be described in this standard (Table 1 and Guide E1525). Ideally, a test organism should: (1) have a toxicological database demonstrating relative sensitivity to a range of contaminants of interest in sediment, (2) have a database for interlaboratory comparisons of procedures (for example, round-robin studies), (3) be in direct contact with sediment, (4) be readily available from culture or through field collection, (5) be easily maintained in the laboratory, (6) be easily identified, (7) be ecologically or economically important, (8) have a broad geographical distribution, be indigenous (either present or historical) to the site being evaluated, or have a niche similar to organisms of concern (for example, similar feeding guild or behavior to the indigenous organisms), (9) be tolerant of a broad range of sediment physico-chemical characteristics (for example, grain size), and (10) be compatible with selected exposure methods and endpoints (Guide E1525). Methods utilizing selected organisms should also be (11) peer reviewed (for example, journal articles) and (12) confirmed with responses with natural populations of benthic organisms.ATL = Atlantic Coast, PAC = Pacific Coast, GOM= Gulf of Mexico1.11.2 Of these criteria (Table 1), a database demonstrating relative sensitivity to contaminants, contact with sediment, ease of culture in the laboratory or availability for field-collection, ease of handling in the laboratory, tolerance to varying sediment physico-chemical characteristics, and confirmation with responses with natural benthic populations were the primary criteria used for selecting A. abdita, E. estuarius, L. plumulosus, and R. abronius for the current edition of this standard for 10-d sediment tests (Annex A1). The species chosen for this method are intimately associated with sediment, due to their tube- dwelling or free-burrowing, and sediment ingesting nature. Amphipods have been used extensively to test the toxicity of marine, estuarine, and freshwater sediments (Swartz et al., 1985 (10); DeWitt et al., 1989 (11); Scott and Redmond, 1989 (12); DeWitt et al., 1992a (6); Schlekat et al., 1992 (13)). The selection of test species for this standard followed the consensus of experts in the field of sediment toxicology who participated in a workshop entitled “Testing Issues for Freshwater and Marine Sediments”. The workshop was sponsored by USEPA Office of Water, Office of Science and Technology, and Office of Research and Development, and was held in Washington, D.C. from 16-18 September 1992 (USEPA, 1992 (15)). Of the candidate species discussed at the workshop, A. abdita, E. estuarius, L. plumulosus, and R. abronius best fulfilled the selection criteria, and presented the availability of a combination of one estuarine and one marine species each for both the Atlantic (the estuarine L. plumulosus and the marine A. abdita ) and Pacific (the estuarine E. estuarius and the marine R. abronius) coasts. Ampelisca abdita is also native to portions of the Gulf of Mexico and San Francisco Bay. Many other organisms that might be appropriate for sediment testing do not now meet these selection criteria because little emphasis has been placed on developing standardized testing procedures for benthic organisms. For example, a fifth species, Grandidierella japonica was not selected because workshop participants felt that the use of this species was not sufficiently broad to warrant standardization of the method. Environment Canada (1992 (5)) has recommended the use of the following amphipod species for sediment toxicity testing: Amphiporeia virginiana, Corophium volutator , Eohaustorius washingtonianus, Foxiphalus xiximeus, and Leptocheirus pinguis. A database similar to those available for A. abdita, E. estuarius, L. plumulosus, and R. abronius must be developed in order for these and other organisms to be included in future editions of this standard.1.11.3 The primary criterion used for selecting L. plumulosus for chronic testing of sediments was that this species is found in both oligohaline and mesohaline regions of estuaries on the East Coast of the United States and is tolerant to a wide range of sediment grain size distribution (USEPA-USACE 2001 (2), Annex Annex A2). This species is easily cultured in the laboratory and has a relatively short generation time (that is, about 24 d at 23 °C, DeWitt et al. 1992a(6)) that makes this species adaptable to chronic testing (Section 12).1.11.4 An important consideration in the selection of specific species for test method development is the existence of information concerning relative sensitivity of the organisms both to single chemicals and complex mixtures. Several studies have evaluated the sensitivities of A. abdita, E. estuarius, L. plumulosus, or R. abronius, either relative to one another, or to other commonly tested estuarine or marine species. For example, the sensitivity of marine amphipods was compared to other species that were used in generating saltwater Water Quality Criteria. Seven amphipod genera, including Ampelisca abdita and Rhepoxynius abronius, were among the test species used to generate saltwater Water Quality Criteria for 12 chemicals. Acute amphipod toxicity data from 4-d water-only tests for each of the 12 chemicals was compared to data for (1) all other species, (2) other benthic species, and (3) other infaunal species. Amphipods were generally of median sensitivity for each comparison. The average percentile rank of amphipods among all species tested was 57 %; among all benthic species, 56 %; and, among all infaunal species, 54 %. Thus, amphipods are not uniquely sensitive relative to all species, benthic species, or even infaunal species (USEPA 1994a (1)). Additional research may be warranted to develop tests using species that are consistently more sensitive than amphipods, thereby offering protection to less sensitive groups.1.11.5 Williams et al. (1986 (16)) compared the sensitivity of the R. abronius 10-d whole sediment test, the oyster embryo (Crassostrea gigas) 48-h abnormality test, and the bacterium (Vibrio fisheri) 1-h luminescence inhibition test (that is, the Microtox2 test) to sediments collected from 46 contaminated sites in Commencement Bay, WA. Rhepoxynius abronius were exposed to whole sediment, while the oyster and bacterium tests were conducted with sediment elutriates and extracts, respectfully. Microtox2 was the most sensitive test, with 63 % of the sites eliciting significant inhibition of luminescence. Significant mortality of R. abronius was observed in 40 % of test sediments, and oyster abnormality occurred in 35 % of sediment elutriates. Complete concordance (that is, sediments that were either toxic or not-toxic in all three tests) was observed in 41 % of the sediments. Possible sources for the lack of concordance at other sites include interspecific differences in sensitivity among test organisms, heterogeneity in contaminant types associated with test sediments, and differences in routes of exposure inherent in each toxicity test. These results highlight the importance of using multiple assays when performing sediment assessments.1.11.6 Several studies have compared the sensitivity of combinations of the four amphipods to sediment contaminants. For example, there are several comparisons between A. abdita and R. abronius, between E. estuarius and R. abronius, and between A. abdita and L. plumulosus. There are fewer examples of direct comparisons between E. estuarius and L. plumulosus , and no examples comparing L. plumulosus and R. abronius. There is some overlap in relative sensitivity from comparison to comparison within each species combination, which appears to indicate that all four species are within the same range of relative sensitivity to contaminated sediments.1.11.6.1 Word et al. (1989 (17)) compared the sensitivity of A. abdita and R. abronius to contaminated sediments in a series of experiments. Both species were tested at 15 °C. Experiments were designed to compare the response of the organism rather than to provide a comparison of the sensitivity of the methods (that is, Ampelisca abdita would normally be tested at 20 °C). Sediments collected from Oakland Harbor, CA, were used for the comparisons. Twenty-six sediments were tested in one comparison, while 5 were tested in the other. Analysis of results using Kruskal Wallace rank sum test for both experiments demonstrated that R. abronius exhibited greater sensitivity to the sediments than A. abdita at 15 °C. Long and Buchman (1989 (18)) also compared the sensitivity of A. abdita and R. abronius to sediments from Oakland Harbor, CA. They also determined that A. abdita showed less sensitivity than R. abronius, but they also showed that A. abdita was less sensitive to sediment grain size factors than R. abronius.1.11.6.2 DeWitt et al. (1989 (11)) compared the sensitivity of E. estuarius and R. abronius to sediment spiked with fluoranthene and field-collected sediment from industrial waterways in Puget Sound, WA, in 10-d tests, and to aqueous cadmium (CdCl2) in a 4-d water-only test. The sensitivity of E. estuarius was from two (to spiked-spiked sediment) to seven (to one Puget Sound, WA, sediment) times less sensitive than R. abronius in sediment tests, and ten times less sensitive to CdCl2 in the water-only test. These results are supported by the findings of Pastorok and Becker (1990 (19)) who found the acute sensitivity of E. estuarius and R. abronius to be generally comparable to each other, and both were more sensitive than Neanthes arenaceodentata (survival and biomass endpoints), Panope generosa (survival), and Dendraster excentricus (survival).1.11.6.3 Leptocheirus plumulosus was as sensitive as the freshwater amphipod Hyalella azteca to an artificially created gradient of sediment contamination when the latter was acclimated to oligohaline salinity (that is, 6 o/oo ; McGee et al., 1993 (20)). DeWitt et al. (1992b (21)) compared the sensitivity of L. plumulosus with three other amphipod species, two mollusks, and one polychaete to highly contaminated sediment collected from Baltimore Harbor, MD, that was serially diluted with clean sediment. Leptocheirus plumulosus was more sensitive than the amphipods Hyalella azteca and Lepidactylus dytiscus and exhibited equal sensitivity with E. estuarius. Schlekat et al. (1995 (22)) describe the results of an interlaboratory comparison of 10-d tests with A. abdita, L. plumulosus and E. estuarius using dilutions of sediments collected from Black Rock Harbor, CT. There was strong agreement among species and laboratories in the ranking of sediment toxicity and the ability to discriminate between toxic and non-toxic sediments.1.11.6.4 Hartwell et al. (2000 (23)) evaluated the response of Leptocheirus plumulosus (10-d survival or growth) to the response of the amphipod Lepidactylus dytiscus (10-d survival or growth), the polychaete Streblospio benedicti (10-d survival or growth), and lettuce germination (Lactuca sativa in 3-d exposure) and observed that L. plumulosus was relatively insensitive compared to the response of either L. dytiscus or S. benedicti in exposures to 4 sediments with elevated metal concentrations.1.11.6.5 Ammonia is a naturally occurring compound in marine sediment that results from the degradation of organic debris. Interstitial ammonia concentrations in test sediment can range from <1 mg/L to in excess of 400 mg/L (Word et al., 1997 (24)). Some benthic infauna show toxicity to ammonia at concentrations of about 20 mg/L (Kohn et al., 1994 (25)). Based on water-only and spiked-sediment experiments with ammonia, threshold limits for test initiation and termination have been established for the L. plumulosus chronic test. Smaller (younger) individuals are more sensitive to ammonia than larger (older) individuals (DeWitt et al., 1997a(7), b (26). Results of a 28-d test indicated that neonates can tolerate very high levels of pore-water ammonia (>300 mg/L total ammonia) for

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5.1 Soil toxicity tests provide information concerning the toxicity and bioavailability of chemicals associated with soils to terrestrial organisms. As important members of the soil fauna, lumbricid earthworms and enchytraeid potworms have a number of characteristics that make them appropriate organisms for use in the assessment of potentially hazardous soils. Earthworms may ingest large quantities of soil, have a close relationship with other soil biomasses (for example, invertebrates, roots, humus, litter, and microorganisms), constitute up to 92 % of the invertebrate biomass of soil, and are important in recycling nutrients (1, 2).4 Enchytraeids contribute up to 5.2 % of soil respiration, constitute the second-highest biomass in many soils (the highest in acid soils in which earthworms are lacking) and effect considerably nutrient cycling and community metabolism (3-5). Earthworms and potworms accumulate and are affected by a variety of organic and inorganic compounds (2-10, 11-14). In addition, earthworms and potworms are important in terrestrial food webs, constituting a food source for a very wide variety of organisms, including birds, mammals, reptiles, amphibians, fish, insects, nematodes, and centipedes (15, 16, 3). A major change in the abundance of soil invertebrates such as lumbricids or enchytraeids, either as a food source or as organisms functioning properly in trophic energy transfer and nutrient cycling, could have serious adverse ecological effects on the entire terrestrial system.5.2 A number of species of lumbricids and enchytraeid worms have been used in field and laboratory investigations in the United States and Europe. Although the sensitivity of various lumbricid species to specific chemicals may vary, from their study of four species of earthworms (including E. fetida) exposed to ten organic compounds representing six classes of chemicals, Neuhauser, et al (7) suggest that the selection of earthworm test species does not affect the assessment of a chemical's toxicity markedly. The sensitivity of various enchytraeid species has not been investigated in a comparable way so far, but ecological importance and practicability reasons favor strongly the selection of a species belonging to the genus Enchytraeus.5.2.1 E. fetida is a species whose natural habitats are those of very high organic matter such as composts and manure piles. It was selected as the test species because it (1) is bred in the laboratory easily; (2) is the earthworm species used most commonly in laboratory experiments (17); (3) has been studied extensively, producing a data pool on the toxicity and bioaccumulation of a variety of compounds (2, 7, 8, 18-23); (4) has been approved for use in toxicity testing by the European Union (EU) and the Organization for Economic Cooperation and Development (OECD); and (5) has been used by the Environmental Protection Agency (EPA) for the toxicity screening of hazardous waste sites (24).5.2.2 The recommended enchytraeid test species is Enchytraeus albidus Henle 1837 (white potworm). E. albidus is one of the biggest (up to 15 mm) species of the oligochaete family Enchytraeidae and it is distributed world-wide (25, 26). E. albidus is found in marine, limnic, and terrestrial habitats, mainly in decaying organic matter (seaweed, compost) and rarely in meadows (4, 26). This broad ecological tolerance and some morphological variations might indicate that there are different races for this species. E. albidus is commercially available, sold as food for fish, can be bred easily in a wide range of organic waste materials and has a short life cycle (33 to 74 days; 27, 28). E. albidus was studied in various tests, which covered a wide range of compounds (28-30). In addition, it is currently under investigation for use in toxicity testing and soil quality assessment by the European Union (EU), the Organization for Economic Cooperation and Development (OECD), and the International Organization for Standardization (ISO). Other species of the genus Enchytraeus are also suitable, for example, E. buchholzi Vejdovsky 1879 or E. crypticus Westheide and Graefe 1992 (see Annex A4). Those species are true soil inhabitants and are smaller in size. Other species of Enchytraeus may be used, but they should be identified clearly and the rationale for their selection should be reported.5.3 Results from soil toxicity tests might be an important consideration when assessing the hazards of materials to terrestrial organisms.5.4 Information might also be obtained on the bioaccumulation of chemicals associated with soil by analysis of animal tissues for the chemicals being monitored. These results are useful for studying the biological availability of chemicals.5.5 The soil toxicity test might be used to determine the temporal or spatial distribution of soil toxicity. Test methods can be used to detect horizontal and vertical gradients in toxicity.5.6 Results of soil toxicity tests could be used to compare the sensitivities of different species.5.7 An understanding of the effect of these parameters on toxicity and bioaccumulation may be gained by varying soil characteristics such as pH, clay content, and organic material.5.8 Results of soil toxicity tests may be useful in helping to predict the effects likely to occur with terrestrial organisms in field situations.5.8.1 Field surveys can be designed to provide either a qualitative or quantitative evaluation of biological effects within a site or among sites.5.8.2 Soil surveys evaluating biological effects are usually part of more comprehensive analyses of biological, chemical, geological, and hydrographic conditions. Statistical correlation can be improved and costs reduced if subsamples of soil for laboratory toxicity tests, geochemical analyses, and community structure are taken simultaneously from the same grab of the same site.5.9 Soil toxicity and bioaccumulation tests can be an important tool for making decisions regarding the extent of remedial action necessary for contaminated terrestrial sites.1.1 This guide covers procedures for obtaining laboratory data to evaluate the adverse effects of contaminants (for example, chemicals or biomolecules) associated with soil to earthworms (Family Lumbricidae) and potworms (Family Enchytraeidae) from soil toxicity or bioaccumulation tests. The methods are designed to assess lethal or sublethal toxic effects on earthworms or bioaccumulation of contaminants in short-term tests (7 to 28 days) or on potworms in short to long-term tests (14 to 42 days) in terrestrial systems. Soils to be tested may be (1) reference soils or potentially toxic site soils; (2) artificial, reference, or site soils spiked with compounds; (3) site soils diluted with reference soils; or (4) site or reference soils diluted with artificial soil. Test procedures are described for the species Eisenia fetida (see Annex A1) and for the species Enchytraeus albidus (see Annex A4). Methods described in this guide may also be useful for conducting soil toxicity tests with other lumbricid and enchytraeid terrestrial species, although modifications may be necessary.1.2 Modification of these procedures might be justified by special needs. The results of tests conducted using atypical procedures may not be comparable to results using this guide. Comparison of results obtained using modified and unmodified versions of these procedures might provide useful information concerning new concepts and procedures for conducting soil toxicity and bioaccumulation tests with terrestrial worms.1.3 The results from field-collected soils used in toxicity tests to determine a spatial or temporal distribution of soil toxicity may be reported in terms of the biological effects on survival or sublethal endpoints (see Section 14). These procedures can be used with appropriate modifications to conduct soil toxicity tests when factors such as temperature, pH, and soil characteristics (for example, particle size, organic matter content, and clay content) are of interest or when there is a need to test such materials as sewage sludge and oils. These methods might also be useful for conducting bioaccumulation tests.1.4 The results of toxicity tests with (1) materials (for example, chemicals or waste mixtures) added experimentally to artificial soil, reference soils, or site soils, (2) site soils diluted with reference soils, and (3) site or reference soils diluted with artificial soil, so as to create a series of concentrations, may be reported in terms of an LC50 (median lethal concentration) and sometimes an EC50 (median effect concentration). Test results may be reported in terms of NOEC (no observed effect concentration), LOEC (lowest observed effect concentration) or as an ECx (concentration where x % reduction of a biological effect occurs. Bioaccumulation test results are reported as the magnitude of contaminant concentration above either the Day 0 tissue baseline analysis or the Day 28 tissues from the negative control or reference soil (that is, 2x, 5x, 10x) (see A3.9).1.5 This guide is arranged as follows:   1  Referenced Documents  2  Terminology  3  Summary of Guide  4   5  Interferences  6  Apparatus  7  Safety Precautions  8  Soil  9  Test Organism 10  Procedure 11  Analytical Methodology 12  Acceptability of Test 13  Calculation of Results 14  Report 15  Annexes     Annex A1. Eisenia fetida     Annex A2. Artificial Soil Composition     Annex A3. Bioaccumulation Testing Utilizing Eisenia fetida   Annex A4. Enchytraeid Reporduction Test (ERT)  References  1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. While some safety considerations are included in this guide, it is beyond the scope of this standard to encompass all safety requirements necessary to conduct soil toxicity tests. Specific precautionary statements are given in Section 8.1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 This test method has been designed to provide data for the mathematical modeling of fire hazard as a means for the evaluation of materials and products and to assist in their research and development.5.1.1 Test Method E1678 is functionally equivalent to NFPA 269-2017.5.2 This test method is used to predict, and subsequently confirm, the lethal toxic potency of smoke produced upon the exposure of a material or product to specific fire test conditions. Confirmation determines whether certain major gaseous toxicants account for the observed toxic effects and lethal toxic potency. If a predicted lethal toxic potency value is not confirmed adequately, indicating a potential for unusual or unexplained toxicity, the lethal toxic potency will need to be investigated using other methodology, such as conducting an experimental determination of the LC50 using the apparatus described. (See X1.3.1 and X1.3.2.)5.3 This test method produces lethal toxic potency values that are appropriate for use in the modeling of both pre-flashover and post-flashover fires. Most fire deaths due to smoke inhalation in the U.S. occur in areas other than the room of fire origin and are caused by fires that have proceeded beyond the room of fire origin. It is assumed that these are flashover fires. Therefore, the principal emphasis is placed on evaluating toxic hazard under these conditions. In post-flashover fires, large concentrations of carbon monoxide results from reduced air supply to the fire plume and other room-scale factors. Bench-scale tests do not have the capacity to simulate these phenomena. The lethal toxic potency values determined in this test method are obtained from fuel/air ratios more representative of pre-flashover, rather than post-flashover conditions. In cases where a pre-flashover fire representation is desired in fire hazard modeling, these LC50 values are appropriate. Lethal toxic potency and carbon monoxide yield values determined in this test method require adjustment for use in modeling of the hazard from post-flashover conditions. (See X1.4.1.)5.4 The lethal toxic potency values determined in this test method have a level of uncertainty in their accuracy when used to predict real-scale toxic potencies. (See X1.4.2.)5.4.1 The accuracy of the bench-scale data for pre-flashover fires has not been established experimentally. The combustion conditions in the apparatus are quite similar to real pre-flashover fires, although the mass burning rate may be higher at the 50 kW/m2 irradiance of the test method.5.4.2 Comparison of the toxicant yields and LC50 (post-flashover) values obtained using this method have been shown in limited tests (1) to reproduce the LC50 values from real-scale, post-flashover fires to within an accuracy of approximately a factor of three. Therefore, LC50 (post-flashover) values differing by less than a factor of three are indistinguishable from each other. (See X1.4.2.)5.5 This test method does not attempt to address the toxicological significance of changes in particulate and aerosol size, smoke transport, distribution, or deposition or changes in the concentration of any smoke constituent as a function of time as may occur in a real fire.5.6 The propensity for smoke from any material to have the same effects on humans in fire situations can be inferred only to the extent that the rat is correlated with humans as a biological system.5.7 This test method does not assess incapacitation. Incapacitation must be inferred from lethal toxic potency values.5.8 The effects of sensory irritation are not addressed by this test method.1.1 This fire-test-response standard covers a means for determining the lethal toxic potency of smoke produced from a material or product ignited while exposed to a radiant heat flux of 50 kW/m2 for 15 min.1.2 This test method is limited to test specimens no larger than 76 mm by 127 mm (3 in. by 5 in.), with a thickness no greater than 51 mm (2 in.). Specimens are intended to be representative of finished materials or products, including composite and combination systems.1.3 Lethal toxic potency values associated with 30-min exposures are predicted using calculations that use combustion atmosphere analytical data for carbon monoxide, carbon dioxide, oxygen (vitiation) and, if present, hydrogen cyanide, hydrogen chloride, and hydrogen bromide. The predictive equations are therefore limited to those materials and products whose smoke toxicity can be attributed to these toxicants. An animal check determines the extent to which additional toxicants contribute to the lethal toxic potency of the smoke.1.4 The values stated in SI units are to be regarded as standard. The values given in parentheses after SI units are provided for information only and are not considered standard.1.5 This standard measures and describes the response of materials, products, or assemblies in response to heat under controlled conditions, but does not by itself incorporate all factors required for fire hazard of fire risk assessment of the materials, products, or assemblies under actual fire conditions.1.6 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations (particularly with regard to the care and use of experimental animals) prior to use. For specific hazards statements, see Section 7 and Note X1.1.1.7 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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5.1 Terrestrial phytotoxicity tests are useful in assessing the effects of environmental samples or specific chemicals as a part of an ecological risk assessment (3-6, 12, 13).5.2 Though inferences regarding higher-order ecological effects (population, community, or landscape) may be made from the results, these tests evaluate responses of individuals of one or more plant species to the test substance.5.3 This guide is applicable for: (a) establishing phytotoxicity of organic and inorganic substances; (b) determining the phytotoxicity of environmental samples; (c) determining the phytotoxicity of sludges and hazardous wastes, (d) assessing the impact of discharge of toxicants to land, and (e) assessing the effectiveness of remediation efforts.1.1 This guide covers practices for conducting plant toxicity tests using terrestrial plant species to determine effects of test substances on plant growth and development. Specific test procedures are presented in accompanying annexes.1.2 Terrestrial plants are vital components of ecological landscapes. The populations and communities of plants influence the distribution and abundance of wildlife. Obviously, plants are the central focus of agriculture, forestry, and rangelands. Toxicity tests conducted under the guidelines and annexes presented herein can provide critical information regarding the effects of chemicals on the establishment and maintenance of terrestrial plant communities.1.3 Toxic substances that prevent or reduce seed germination can have immediate and large impacts to crops. In natural systems, many desired species may be sensitive, while other species are tolerant. Such selective pressure can result in changes in species diversity, population dynamics, and community structure that may be considered undesirable. Similarly, toxic substances may impair the growth and development of seedlings resulting in decreased plant populations, decreased competitive abilities, reduced reproductive capacity, and lowered crop yield. For the purposes of this guide, test substances include pesticides, industrial chemicals, sludges, metals or metalloids, and hazardous wastes that could be added to soil. It also includes environmental samples that may have had any of these test substances incorporated into soil.1.4 Terrestrial plants range from annuals, capable of completing a life-cycle in as little as a few weeks, to long-lived perennials that grow and reproduce for several hundreds of years. Procedures to evaluate chemical effects on plants range from short-term measures of physiological responses (for example, chlorophyll fluorescence) to field studies of trees over several years. Research and development of standardized plant tests have emphasized three categories of tests: (1) short-term, physiological endpoints (that is, biomarkers); (2) short-term tests conducted during the early stages of plant growth with several endpoints related to survival, growth, and development; and (3) life-cycle toxicity tests that emphasize reproductive success.1.5 This guide is arranged by sections as follows:Section Title1 2 Referenced Documents3 Terminology4 Summary of Phytotoxicity Tests5 6 Apparatus7 Test Material8 Hazards9 Test Organisms10 Sample Handling and Storage11 Calibration and Standardization12 Test Conditions13 Interference and Limitations14 Quality Assurance and Quality Control15 Calculations and Interpretation of Results16 Precision and Bias1.6 The values stated in SI units are to be regarded as standard. No other units of measurement are included in this standard.1.7 This standard does not purport to address all of the safety concerns, if any, associated with its use. It is the responsibility of the user of this standard to establish appropriate safety, health, and environmental practices and determine the applicability of regulatory limitations prior to use. Specific precautionary statements are given in Section 8.1.8 This international standard was developed in accordance with internationally recognized principles on standardization established in the Decision on Principles for the Development of International Standards, Guides and Recommendations issued by the World Trade Organization Technical Barriers to Trade (TBT) Committee.

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